Impaired proinsulin-to-insulin processing in pancreatic β-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. ^1,2), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. ^3–8); nonetheless, the role of specific SL species in β-cell function and demise is unclear. Here we define the lipid signature of T2D-associated β-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. β-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL–protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum–Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in β-cell function and T2D-associated β-cell failure.

Several antitumor therapies work by increasing reactive oxygen species (ROS) within the tumor micromilieu. Here, we reveal that L-plastin (LPL), an established tumor marker, is reversibly regulated by ROS-induced thiol oxidation on Cys101, which forms a disulfide bridge with Cys42. LPL reduction is mediated by the Thioredoxin1 (TRX1) system, as shown by TRX1 trapping, TRX1 knockdown and blockade of Thioredoxin1 reductase (TRXR1) with auranofin. LPL oxidation diminishes its actin-bundling capacity. Ratiometric imaging using an LPL-roGFP-Orp1 fusion protein and a dimedone-based proximity ligation assay (PLA) reveal that LPL oxidation occurs primarily in actin-based cellular extrusions and strongly inhibits cell spreading and filopodial extension formation in tumor cells. This effect is accompanied by decreased tumor cell migration, invasion and extracellular matrix (ECM) degradation. Since LPL oxidation occurs following treatment of tumors with auranofin or γ-irradiation, it may be a molecular mechanism contributing to the effectiveness of tumor treatment with redox-altering therapies.

Significance: Human hCDC14A phosphatase has been shown to regulate cell migration and adhesion by modulating actin dynamics. Here we identify the potent tumor suppressor eplin as an hCDC14A substrate. Through dephosphorylation of serine residues 362 and 604 of eplin, hCDC14A promotes F-actin bundling and counteracts EGF-induced membrane ruffling. Cells lacking hCDC14A activity or eplin expression showed reduced E-cadherin levels and β-catenin retention at cell–cell junctions and formed more invasive colonies than wild-type cells. Expression of a phospho-inhibitory, but not a phospho-mimetic, eplin mutant rescued these phenotypes. Database analysis indicated that hCDC14A and eplin are frequently inactivated in human colorectal cancer and this is associated with poor prognosis. Therefore, the hCDC14A and eplin axis plays an important role in limiting tumor malignancy.

The fatty acid 2-hydroxylase (FA2H) is essential for synthesis of 2-hydroxylated fatty acids in myelinating and other cells, and deficiency of this enzyme causes a complicated form of hereditary spastic paraplegia also known as fatty acid hydroxylase-associated neurodegeneration. Despite its important role in sphingolipid metabolism, regulation of FA2H and its interaction with other proteins involved in the same or other metabolic pathways is poorly understood. To identify potential interaction partners of the enzyme, quantitative mass spectrometry using stable isotope labeling of cells was combined with formaldehyde cross-linking and proximity biotinylation, respectively. Besides other enzymes involved in sphingolipid synthesis and intermembrane transfer of ceramide, and putative redox partners of FA2H, progesterone receptor membrane component-1 (PGRMC1) and PGRMC2 were identified as putative interaction partners. These two related heme-binding proteins are known to regulate several cytochrome P450 enzymes. Bimolecular fluorescence complementation experiments confirmed the interaction of FA2H with PGRMC1. Moreover, the PGRMC1 inhibitor AG-205 significantly reduced synthesis of hydroxylated ceramide and glucosylceramide in FA2H-expressing cells. This suggests that PGRMC1 may regulate FA2H activity, possibly through its heme chaperone activity.

Niemann-Pick type C disease (NPCD) is a lysosomal storage disorder caused by mutations in the NPC1 gene. The most affected tissues are the central nervous system and liver, and while significant efforts have been made to understand its neurological component, the pathophysiology of the liver damage remains unclear. In this study, hepatocytes derived from wild type and Npc1^−/− mice were analyzed by mass spectrometry (MS)-based proteomics in conjunction with bioinformatic analysis. We identified 3832 proteins: 416 proteins had a p-value smaller than 0.05, of which 37% (n = 155) were considered differentially expressed proteins (DEPs), 149 of them were considered upregulated, and 6 were considered downregulated. We focused the analysis on pathways related to NPC pathogenic mechanisms, finding that the most significant changes in expression levels occur in proteins that function in the pathways of liver damage, lipid metabolism, and inflammation. Moreover, in the group of DEPs, 30% (n = 47) were identified as lysosomal proteins and 7% (n = 10) were identified as mitochondrial proteins. Importantly, we found that lysosomal DEPs, including CTSB/D/Z, LIPA, DPP7 and GLMP, and mitocondrial DEPs, AKR1B10, and VAT1 had been connected with liver fibrosis, damage, and steatosis in previous studies, validiting our dataset. Our study found potential therapeutic targets for the treatment of liver damage in NPCD.